U.S. patent number 3,873,680 [Application Number 05/285,079] was granted by the patent office on 1975-03-25 for mercaptan and thioketal complexes of technetium 99m for diagnostic scanning.
This patent grant is currently assigned to Minnesota Mining and Manufacturing Company. Invention is credited to Theodore F. Bolles, Richard A. Jackson.
United States Patent |
3,873,680 |
Jackson , et al. |
March 25, 1975 |
Mercaptan and thioketal complexes of technetium 99M for diagnostic
scanning
Abstract
Chemical complexes containing a radioactive technetium isotope
are liver specific when the complexing agent is a lipophilic
mercaptan or thioketal. To permit excretion of the complex from the
liver into the bile, the lipophilic mercaptan contains a polar
group which moderates this lipophilic property. The complexes can
be made by reducing pertechnetate ion and reacting the reduced
technetium species with the mercaptan or thioketal. The complexes
are normally used in a biologically sterile substantially isotonic
aqueous medium.
Inventors: |
Jackson; Richard A. (White Bear
Lake, MN), Bolles; Theodore F. (Woodbury, MN) |
Assignee: |
Minnesota Mining and Manufacturing
Company (St. Paul, MN)
|
Family
ID: |
23092651 |
Appl.
No.: |
05/285,079 |
Filed: |
August 30, 1972 |
Current U.S.
Class: |
424/1.65; 534/14;
250/303; 424/1.77 |
Current CPC
Class: |
A61K
51/04 (20130101); C07C 323/00 (20130101); A61K
2123/00 (20130101) |
Current International
Class: |
A61K
51/02 (20060101); A61K 51/04 (20060101); A61k
027/04 () |
Field of
Search: |
;424/1 ;23/23B ;250/303
;252/31.1R ;260/69R,632R,429R |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Padget; Benjamin R.
Attorney, Agent or Firm: Alexander, Sell, Steldt &
DeLaHunt
Claims
What is claimed is:
1. A process for making a reaction product which can be liver
specific in vivo comprising the steps of:
a. reducing a solution comprising at least 0.01 but less than 500
millicuries per milliliter of 99m-pertechnetate ion until more than
50 mole % of said 99m-pertechnetate ion has been reduced to a
99m-technetium species having an oxidation state greater than zero
but less than +7,
b. reacting said 99m-technetium species with an excess, regardless
of the stoichiometry of the resulting product, of a
sulfur-containing compound selected from the group consisting
of:
A. a lipophilic compound of the formula
HS -- CR.sup.1 R.sup.2 --.sub.n X
wherein R.sup.1 and R.sup.2 are the same or different in each
repeating unit and are selected from the group consisting of
hydrogen, lower alkyl, and --SH,
n is an integer ranging from 1 to about 6, and
X is a mammalian blood-solubilizing polar group which only
partially detracts from the liver specific and lipophilic
properties of the said resulting reaction product;
B. a lipophilic compound of the formula
HS -- A -- X
where
A is a cycloaliphatic or heterocyclic aliphatic ring and X is as
defined previously;
C. a lipophilic thioenol or thioketal substituted on the thioketal-
or thioenol-carbon with a water solubilizing group and, on the
carbon alpha to the thioketal- or thioenol carbon, wtih a
lipophilic group; and
D. a carbocyclic or heterocyclic aromatic carboxylic, sulfonic, or
phosphonic acid bearing a mercapto group substituted more remotely
than ortho to the carboxylic, sulfonic, or phosphonic acid
group.
2. A process according to claim 1 wherein said sulfur-containing
compound is itself used to chemically reduce the 99m pertechnetate
compound, thereby combining said reducing step and said reacting
step.
3. A process according to claim 1 wherein said sulfur-containing
compound is selected from the group consisting of
A. hs -- cr.sup.3 r.sup.4 --.sub.n X
wherein
R.sup.3 and R.sup.4 are the same or different and are selected from
the group consisting of hydrogen and methyl,
n is an integer ranging from 1 to 5, and
X is selected from the group consisting of
--OH;--NR.sup.2.sub.2,
where
R.sup.2 is hydrogen or methyl; ##SPC6##
where
M.sup.+ is a pharmaceutically acceptable cation; -- N(CH.sub.3) --
NH--COCH.sub.3 ; -- NH--COCH.sub.3 ; -- N(CH.sub.3) NH.sub.2 ;
##SPC7##
where
M.sup.+ is as defined previously,
Z is hydrogen or mercapto, and
y is a positive integer less than 31; and ##SPC8##
where
Ar is an aromatic nucleus, and
X is as defined in claim 1;
B. the compound ##SPC9##
where
X.sup.1 is a lipophilic group; and
C. a meta- or para-mercaptobenzoic acid.
4. A process according to claim 3 wherein said sulfur-containing
compound is selected from the group consisting of
N-acetyl-penicillamine,
2-mercaptoethanol,
3-mercaptopropanol,
4-mercaptobutanol
4-mercapto-2-methyl-2-butanol,
2-mercaptoethylamine,
6,8-dihydrothioctic acid,
alpha-thio-2-furan pyruvic acid,
p-mercaptobenzoic acid, and mixtures thereof.
5. A process according to claim 1 wherein said reducing step is
carried through the use of a reducing agent selected from the group
consisting of a tin (II) salt, an iron (II) salt, a Cu(I)/Cu(II)
couple, and combinations thereof.
6. A process according to claim 2 wherein said process is
acid-catalyzed.
7. A process according to claim 1 wherein said 99m-pertechnetate
ion is derived from the dissociation of a salt of the formula
M.sup.+.sup.x (TcO.sub.4.sup.-).sub.x
wherein
M.sup.+.sup.x is a pharmaceutically acceptable cation having a
valence of x, and
x is a positive integer less than 4.
8. A process according to claim 1 wherein 0.1 - 10 ml. of an
aqueous solution of the reaction product obtained from step (b) is
injected into the blood stream of a living mammal.
9. A process according to claim 1 wherein X is selected such that
the natural logarithm of the partition coefficient of the resulting
reaction product, expressed as the natural logarithm of the ratio
of the activity in water to the activity in n-octyl alcohol, has
the following values:
less than 2 at a pH of 5 to 6
less than 5 at a pH of 7, and
less than 9 at a pH of 8.
10. A process according to claim 9 wherein said natural logarithm
of said ratio is at least -2 at a pH of 6 and is less than 8 at a
pH of 8.
11. A process according to claim 8 wherein said excess of
sulfur-containing compound does not exceed 25% of the LD 50 in
rodents and in at least one other species and wherein said solution
is substantially isotonic.
12. A liver specific 99mTc/6,8-dihydrothioctic acid complex, said
complex being characterized by the following R.sub.f values
determined by thin layer chromatography on a 100 micron-thick,
unactivated, neutral pH silica gel chromatogram sheet, wherein the
silica gel of said chromatogram sheet is bound together with
polyvinyl alcohol:
R.sub.f = 0, when said chromatogram is developed with anhydrous
acetone;
R.sub.f = 0.66, when said chromatogram developed with a solvent
system consisting of ethanol:water:concentrated ammonium hydroxide
in the volume/volume/volume ratio of 95:17:16.
13. A liver specific 99mTc/4-mercaptobutanol complex, said complex
being characterized by the following R.sub.f values determined by
thin layer chromatography on a 100 micron-thick, unactivated,
neutral pH silica gel chromatogram sheet, wherein the silica gel of
said chromatogram sheet is bound together with polyvinyl
alcohol:
R.sub.f = 0, when said chromatogram is developed with anhydrous
acetone;
R.sub.f = 0.69, when said chromatogram is developed with anhydrous
methanol.
Description
FIELD OF THE INVENTION
This invention relates to chemical complexes of technetium. An
aspect of this invention relates to chemical complexes of the
radioactive, metastable isotope technetium-99m (Tc-99m). A further
aspect of this invention relates to complexes of technetium-99m
wherein the complexing agent is a lipophilic sulfur-containing
compound such as a mono- or poly-mercaptan or a mono- or
polythioketal. A further aspect of this invention relates to a
process for producing the chemical complex and a preferred
biologically sterile substantially isotonic medium containing the
complex. Still another aspect of this invention relates to the use
of the processes and products of this invention in studies of liver
and/or gallbladder function.
DESCRIPTION OF THE PRIOR ART
The art of radiochemistry has found many applications in the fields
of medicine and biology. It has long been known that the
introduction into an organism of compounds containing (or "labeled"
with) a radioisotope can provide insight into the anatomy and
physiology of the organism. These compounds, generally referred to
as radiopharmaceuticals, are particularly useful in diagnostic
techniques which involve studying the structure or function of
various internal organs, e.g. the brain, with radiation detection
means. For diagnostic work, isotopes with a short half life and an
emission spectrum rich in gamma rays (as opposed to beta particles)
are preferred.
The metastable isotope Tc-99m has a 6 hour half-life and an
emission spectrum, 99% gamma radiation at 140 KeV, which is
extremely well suited for techniques of diagnostic nuclear
medicine. Thus, Tc-99m has a high specific activity, 5.28 .times.
10.sup.9 millicuries per gram (mc/g), and a conveniently rapid rate
of decay; whereas its daughter product, Tc-99, has a specific
activity which is almost nine orders or magnitude lower and a half
life which is roughly eight orders of magnitude longer. For the
organism being studied or diagnosed, the slow rate of decay from
the relatively stable, low specific activity Tc-99 to its
degradation product (ruthenium) would not normally produce any
hazardous amounts of radiation, regardless of the biological means
or route of elimination of a Tc-99m radiopharmaceutical. For the
researcher or clinician, the emission spectrum of Tc-99m can
provide high levels of accuracy in radiodiognostic measurements and
calculations. In recent years, Tc-99m has become readily available
in hospitals through the use of selective elution from a so-called
molybdenum-99 (Mo-99) generator. The isotope Mo-99 produces Tc-99M
as a radioactive decay product.
Although Tc-99m compounds would appear to be ideal
radiopharmaceuticals for diagnostic use, providing or selecting Tc
compounds or complexes with a view toward organ specificity and
tolerable levels of toxicity is a complex task. Obviously,
compounds with a very low LD 50 are undesirable for human or
veterinary use, even in the small amounts called for by diagnostic
work. Compounds with insufficient in vivo stability may be poor
diagnostic tools, since radioactive ions or other chemical species
with insufficient or undesired organ specificity may be liberated.
Stable compounds which become distributed generally throughout the
organism, despite their stability, or which do not reach a desired
destination in the organism are also poorly suited for many studies
of organ function or structure, e.g. liver and gallbladder studies.
For these studies of organ function, compounds which are specific
to an organ, but which are not excreted by it (or if excreted, are
easily reabsorbed) are also poor candidates.
The problem of selecting or preparing a liver specific
radiopharmaceutical for liver function studies is particularly
difficult. Both the liver and the kidney are capable of removing
various types of compounds from the body -- ultimately through
excretion in feces and urine, respectively. Any radiopharmaceutical
used for this purpose should ideally have 100% liver specificity
and 0% kidney specificity. Ideally, the compound should also be
readily excreted by the liver into the bile. A number of biological
and chemical factors must be considered and brought under control
before the desired organ specificity and route of excretion can be
obtained. For example, some Tc compounds are easily transformed to
TcO.sub.2, which may lodge in the liver but may not be easily
excreted.
Technetium-99m compounds have been used in brain or other organ
scanning. For example, Tc-sulfur colloid can be used for liver
scanning. Organ scanning is useful for studies of organ structure,
but gives little insight into organ function. Representative of the
literature relating to the radiopharmacology of Tc-99m compounds
are the following articles:
Larson et al, J. Nuclear Medicine, 7:8:7 (1966), relating to
Tc-99m-colloid preparation for photoscanning, and
Tubis et al, International Journal of Applied Radiation and
Isotopes, V. 19, 835 (1968), relating to Tc-99m labeled cystine,
methionine, and a synthetic polypeptide and their distribution in
mice.
Compared to the common transition metals, very little is known
about the chemistry of technetium. Technetium belongs to Group
VII-B of the Periodic Table; its chemistry bears a superficial
resemblance to manganese but tends to be more similar to the higher
member of the Group, rhenium. Technetium can apparently exist in a
range of oxidation states, including +7 (e.g. pertechnetate) and
several lower oxidation states, some of which are difficult to
characterize and/or are relatively unstable. In spectrophotometric
determinations of technetium, the element has been complexed with
toluene-3,4-dithiol, thioglycolic acid, and thiocyanates. See
Miller et al, Anal. Chem., Page 404 (1961) and Page 1429 (Oct.,
1960), and Crouthamel, Anal. Chem., page 1756 (Dec., 1957).
Accordingly, this invention contemplates providing complexes of
Tc-99m which have sufficient in vivo stability and a sufficiently
high LD 50 for use in humans or animals and which preferably
are:
Removed from the blood or other vital organs or tissues by the
liver rather than by, for example, the kidneys or the lungs,
Concentrated in the liver at a high rate,
Concentrated in other organs or tissues -- particularly organs or
tissues in close proximity to the liver -- at a very low or
negligible rate,
Retained for a short period of time by the liver and secreted into
the bile,
Removed ultimately from the body by means of a route passing
through the gallbladder and intestines to the feces, and
Eliminated from the body by alternative routes, (e.g. kidney --
bladder -- urine) to a minor, preferably negligible, extent.
This invention further contemplates means and methods whereby Tc
complexes can be most efficiently produced and utilized for liver
function studies.
BRIEF SUMMARY OF THE INVENTION
Briefly, this invention involves reducing an appropriate amount of
radioactive pertechnetate ion (.sup.99m TcO.sub.4.sup.-) until a
major amount of the pertechnetate ion has been reduced to a
technetium species having an oxidation state greater than 0 but
less than +7 and then reacting this technetium species with an
excess of one of the subsequently described sulfur-containing
complexing agents. The resulting Tc-99m complex is suitable for
injection into the blood stream of a mammal when dissolved or
dispersed in a biologically sterile aqueous medium substantially
isotonic with mammalian body fluids. The reduction step can be
carried out chemically through acid catalysis if the complexing
agent is also a reducing agent, at least when the complexing agent
is present in large excess, as will normally be the case.
Preferably, however, reduction is achieved through the use of an
additional reducing agent such as a tin (II) salt, an iron (II)
salt, a copper (I)/copper (II) couple, or a combination of two or
more of these agents.
A meaningful picture of liver function will be obtained by
measuring the radioactivity emitted from the liver, gallbladder,
intestines, and feces of the organism or patient being studied. It
will generally not be necessary to monitor the radioactivity for
more than about 24 hours after the injection, and 12 hours of
monitoring can be fully sufficient.
Products produced by the previously described process can have the
desired liver specificity if the complexing agent belongs to one of
the following four classes of sulfur-containing compounds:
A. a lipophilic compound of the formula
HS(CR.sup.1 R.sup.2).sub.n X
wherein
R.sup.1 and R.sup.2 are the same or different in each repeating
unit and can be hydrogen, lower alkyl, or mercapto,
n is an integer ranging from 1 to about 6, and
X is a mammalian blood-solubilizing polar group which only
partially reduces the lipophilic properties of the Tc complex,
whereby the complex is readily taken up by the liver in preference
to the kidney, despite the X group;
B. a lipophilic compound of the formula
HS-A-X
wherein
A is a cycloaliphatic or heterocyclic aliphatic ring, and
X is as defined previously;
C. a lipophilic thioenol or thioketal substituted on the thioketal-
or thioenol-carbon with a water solubilizing group and, on the
carbon alpha to the thioketal- or thioenol-carbon, with a
lipophilic group; and
D. a carbocyclic or heterocyclic aromatic carboxylic, sulfonic, or
phosphonic acid bearing a mercapto group substituted more remotely
than ortho to the carboxylic, sulfonic, or phosphonic acid
group.
DETAILED DESCRIPTION OF THE INVENTION
As pointed out previously, complexing agents elected for use in
this invention are capable of providing a Tc-99m liver specific
compound which is suitable for inclusion in injectable media
substantially isotonic with mammalian body fluids, which in vivo is
rapidly removed from the blood or other tissues by the liver, is
excreted by the liver into the bile, and therefore into the gut
(directly or through the gall-bladder), is excreted completely from
the gut into the feces, and is not, to any great extent, removed by
other organs nor reabsorbed once excreted by the liver. Thus,
selection of complexing agents according to this invention involves
weighing a combination of chemical and biological criteria.
Chemical considerations alone do not insure that the compound will
have a liver specificity, compatibility with blood or other
mammalian body fluids, or the like. The complexing agent,
furthermore, cannot be considered in a vacuum apart from the
properties of the complex which will result after reaction with
technetium. For example, some complexing agents are lipophilic and
are eliminated by the liver, but tend to form stable, insoluble
complexes with technetium which would not be secreted into the
bile.
Despite all these difficulties of predicting utility of potential
complexing agents from chemical criteria alone, it has now been
found that partition coefficient data of the Tc complexes provides
considerable insight into the proper balance of lipophilic and
hydrophilic properties characteristic of a good liver specific
complex. These partition coefficient studies involve plotting pH
vs. the natural logarithm of the ratio of the activity in water to
the activity in normal octyl alcohol, hereinafter referred to as
1n(a.sub.w /a.sub.o). In the pH range of 5 to 8, the complex should
not be so lipophilic as to have an 1n(a.sub.w /a.sub.o) of less
than -2, particularly at the higher pH levels. On the other hand,
the hydrophilicity must also be kept within limits, as the
following table illustrates:
pH Partition Coefficient = ln(a.sub.w /a.sub.o)
______________________________________ Maximum Preferred
______________________________________ 5 2 < 1.5 6 2 < 1.5 7
5 < 5 8 9 < 8 ______________________________________
Compounds or complexing agents which are particularly suitable for
producing technetium complexes with the desired partition
coefficient and liver specificity characteristics have already been
described in general terms and are typically exemplified by
meta- and para-mercapto benzoic acid and their derivatives, the
thioenol compound
D.sup.1 --CH=C(SH)COOH,
where
X.sup.1 is a lipophilic group (e.g. a furan moiety), and
aliphatic mercaptans containing a water-solubilizing group,
typically of the following structural formula:
HS -- CR.sup.3 R.sup.4 --.sub.n X (1)
wherein
R.sup.3 and R.sup.4 are hydrogen or alkyl, preferably methyl,
n is an integer from 1 to 5, preferably 2 to 4 when R.sup.3 and
R.sup.4 are hydrogen, and
X can be one of the following groups:
hydroxyl; primary, secondary or tertiary amine, the secondary and
tertiary amines being preferably substituted with lower aliphatic
groups (the term "lower" is used to mean groups with less than 7
carbon atoms);
an alpha-amino acid moiety, provided that the radical -- CR.sup.3
R.sup.4 --.sub.n contains at least three carbon atoms in any
branched or straight chain configuration and/or provided that the
carboxylic acid or amine function of the alpha-amino acid moiety is
blocked or substituted with an ionization-preventing radical, e.g.
by acylation of the amine;
a carboxylic acid group, provided the -- CR.sup.3 R.sup.4 --.sub.n
radical contains at least three carbon atoms;
an aliphatic chain (preferably containing less than 31 carbon
atoms) terminated with a carboxylic acid group and optionally
substituted with an additional mercapto (SH) group;
an N-aliphatic substituted hydrazine moiety, wherein the terminal
--NH.sub.2 is optionally converted to an amide;
an amido group, preferably acetamide or other lower aliphatic
amide; and
phenolic groups, provided that the phenolic nucleus contains at
least one additional water-solubilizing group (e.g. one of the
previously described "X" groups) in addition to the phenolic
hydroxyl.
Thus, in structural formula (I), examples of suitable X radicals
include
--NH.sub.2 ; --NH(CH.sub.3); --N(CH.sub.3).sub.2 ; ##SPC1##
where
Q is a blocking group such as N-acetyl, and
M is hydrogen or a pharmaceutically acceptable cation;
--NH--COCH.sub.3 ; --N(CH.sub.3)--NH--COCH.sub.3 ;
--N(CH.sub.3)NH.sub.2 ;
and ##SPC2##
where
y is a positive integer less than 31, preferably less than 6,
M is as defined previously, and
Z is hydrogen or, preferably, a mercapto group.
As will be apparent from the foregoing examples, both ionizable
(including protonatable) and nonionic X groups can be used, but
groups capable of forming zwitterions or other stable internal salt
groups are not preferred. Apparently, zwitterions may have a
tendency to be removed by the kidney, while aliphatic amines,
aliphatic alcohols, and carboxylic acids are less likely to have
this tendency.
Typical preferred members of the previously described classes of
compounds include: N-acetyl-penicillamine (which is greatly
preferred to penicillamine itself, since the amine group is
blocked), the omega-mercaptoalkanols (2-mercaptoethanol,
3-mercaptopropanol, and 4-mercaptobutanol),
4-mercapto-2-methyl-2-butanol, p-mercaptobenzoic acid,
2-mercaptoethylamine, 6,8-dihydrothioctic acid, and
alpha-thio-2-furan pyruvic acid.
The term "lipophilic" is not an absolute term, but like the terms
"hydrophilic," "aqueous," "dispersion," etc., it has well
understood meaning; see the "DEFINITIONS" in Columns 2 and 3 of
U.S. Pat. No. 3,069,370 (Jansen et al.), issued Dec. 18, 1962.
The term "substantially isotonic with mammalian body fluids," as
used herein, denotes the situation obtained when the osmotic
pressure exerted by the solution in question is sufficiently
similar, as compared to a body fluid such as blood, so that no
dangerous hypo- or hypertonic condition results in the patient or
test animal when 0.1 ml (in the case of a mouse) or up to 10 ml (in
the case of a human) of the solution is injected into the patient's
or animal's bloodstream.
The exact mechanism by which the complexing agents used in this
invention become chemically linked to technetium is difficult to
determine. It appears that the Tc-99m should be present primarily
in an oxidation state of at least about +3 but not more than +6.
This oxidation state can be conveniently obtained by reducing
99m-pertechnetate, a relatively stable +7 technetium species. The
reduced species can co-ordinate with one or two sulfur atoms which
are in the form of mercaptan groups of the like. Complexing Tc with
polymercaptans capable of chelating the Tc is preferred for
stability; provided, at least one of the previously defined
solubilizing (X) groups is present. Non-solubilized chelates can be
liver specific but are not necessarily excreted into the bile;
therefore, they are better suited for liver structure studies than
liver function studies.
The amount of Tc-99m needed to produce an amount of
radiopharmaceutical suitable for most diagnostic or research uses
is extremely small and is generally in the range of about 0.01
millicuries per milliliter (mc/ml) of 99m pertechnetate solution up
to about 500 mc per ml of such solution. Only about 0.03 .times.
10.sup.-.sup.10 gram of 99m-pertechnetate dissolved in a milliliter
of aqueous medium (e.g. isotonic saline) is needed to provide 0.01
mc/ml, and less than 100 .times. 10.sup.-.sup.10 gram of
99m-pertechnetate per milliliter of solution provides enough
radioactivity for most uses. Due to the short half-life of the
Tc-99m, it is preferred to prepare small batches of
99m-pertechnetate solution for immediate use. Batches as small as
0.1 ml can be adequate for animal studies (e.g. for injection in
mice) and batches as large as 50 ml are convenient for one or more
injections in one or a group of human patients. In any event, it
would be a rare situation that required more than about 100 .times.
10.sup.-.sup.10 gram (i.e. about 10.sup.-.sup.10 gram-atoms) of
Tc99m as pertechnetate ion to produce a few ml of
radiopharmaceutical, regardless of stoichiometry of the Tc complex.
It is preferred to provide enough complexing agent (ordinarily at
least 5 .times. 10.sup.-.sup.9 moles per milliliter of reaction
mixture) to have an excess over stoichiometry with respect to the
Tc99m in the reaction mixture. A large excess of complexing agent
(e.g. 0.5 - 1,000 micromoles of complexing agent per ml of reaction
mixture) can be desirable, particularly when the complexing agent
itself serves as the means for reducing the oxidation state of
pertechnetate.
Combinations of complexing agents or reducing agents can be used to
achieve desired effects such as lower toxicity or greater chemical
or biological stability.
The Tc-99m used in this invention is obtainable from a Mo-99
generator in the conventional manner. Eluting or "milking" the
generator with an aqueous medium will provide the 99m-pertechnetate
solution in the form of M.sup.+.sup.x (99mTcO.sub.4.sup.-).sub.x,
where M.sup.+.sup.x is a pharmaceutically acceptable cation such as
a proton, an alkali metal ion, an ammonium ion, or the like, and x
is a positive integer less than 4. Typically, the aqueous elution
medium is a saline solution, which provides sodium
99m-pertechnetate.
The pertechnetate ion can be reduced chemically or electrolytically
to a lower oxidation state of technetium, preferably by reaction
with an oxidizable low valence metal salt such as a tin (II) salt
(e.g. SnCl.sub.2), an iron (II) salt (e.g. a ferrous salt/ascorbic
acid medium), a Cu(I)/Cu(II) couple, a combination thereof, or
other chemical reducing agents such as mercaptans, metal hydrides,
thiosulfates, hypophosphites, bromides, iodides, etc. A
particularly suitable means for providing the reducing agent and
complexing agent is to pre-formulate a radiopharmaceutical kit for
use with the Mo-99 generator. For example, 0.1 preferably at least
0.5) to 10 ml. of a solution containing about 0.5 to about 1000
.mu. mole/ml of complexing agent and a suitable amount, e.g. 0.01 -
100 micromoles/ml of reducing agent can be hermetically and
aseptically sealed in separate vials or the same vial. A
preservative such as benzyl alcohol is optionally included in the
contents of the vial. The solution in the vial is preferably
substantially isotonic with mammalian body fluids, e.g. human
blood. The contents of the vial can be combined with the
pertechnetate-containing, substantially isotonic eluate, mild heat
can be applied if necessary to the combined solutions to achieve
the reduction and Tc-complex formation, and the resulting
radio-pharmaceutical can then be injected into the blood stream of
the patient or test animal. Radioactivity measurements are made in
the conventional manner for a period from the time of injection
until about 24 hours afterwards, depending on the nature of the
study or diagnosis. Most studies call for at least one half hour of
post injection radioactive measurements. These measurements can be
corrected for decay in the usual manner and studied with a view
toward obtaining a picture of liver or gallbladder function. If the
patient or test animal is placed on an appropriately controlled
diet prior to liver uptake of the Tc-99m radiopharmaceutical, the
bile, which will contain Tc-99m, will be introduced into the gut by
the gallbladder, thus providing an opportunity for
cholecystography. The gallbladder will not concentrate the Tc-99m
if the patient has ingested fatty foods prior to and after the
injection.
The amount of complexing agent injected into a test animal or human
patient should preferably be less than 25% (e.g. less than 10%) of
the LD 50 in mg per kg of body weight, though higher amounts are
permissible in veterinary medicine. Typical LD 50's (determined in
rodents and at least one other species) for preferred complexing
agents of this invention ranged from about 20 to about 500 mg per
kg of body weight.
As pointed out in the previous discussion regarding partition
coefficients, a balance of lipophilic and hydrophilic properties is
preferred for the Tc-complexing agents of this invention. Aliphatic
mercapto alcohols, amines, and amides appear to provide this
balance through the lipophilic contribution of the aliphatic
portion of the molecule and the hydrophilic contribution of the
amine or hydroxyl radical. (After complexing with Tc, the mercapto
group is probably not sufficiently free to affect the solubility
characteristics of the complex.) Aliphatic mercapto amines and
amides can be derived from a hydrazine nucleus, so to speak, as in
the case of N-methyl,N-(2-mercapto ethyl)-N'-aceto hydrazine and
N-methyl,N-(2-mercapto ethyl)hydrazine.
An especially preferred class of complexing agents includes the
mercapto-substituted aliphatic carboxylic acids and salts thereof.
The mercapto group can be substituted on a primary, secondary, or
tertiary carbon atom, as exemplified by 6,8-dihydrothioctic acid
(HS--C.sub.2 H.sub.4 --CH(SH)--C.sub.4 H.sub.8 COOH) thiolactic
acid, HS--CH(CH.sub.3)COOH, and N-acetyl penicillamine. Alpha-amino
acids (such as penicillamine) having sufficient aliphatic character
are marginally operative as complexing agents in this invention,
but it is preferred to block the alpha-amino group with, for
example, an N-acetyl substituent. Thus, cysteine-Tc lacks
sufficient liver specificity and is not useful in this
invention.
When optical isomerism is possible, as in the case of
dihydrothioctic acid, DL-racemic mixtures are fully operative in
the invention and are easier to synthesize than the individual
isomers. If desired, however, racemic mixtures can be resolved by
conventional techniques.
A complexing agent of this invention preferably contains one of the
aforementioned hydrophilic groups (amine, amide, alcohol, acid,
ester, salt, etc.) but need not be water soluble. Dispersible, but
substantially water insoluble, complexing agents can be dispersed
in water by conventional techniques such as agitation. For example,
higher hydrocarbon groups or chains in the complexing agent (e.g. 6
- 31-carbon saturated or unsaturated aliphatic chains, terminated
with a carboxylic acid group or the like), though sharply reducing
or preventing the water solubility of the complexing agent, would
nevertheless permit the formation of stable aqueous suspensions or
emulsions.
Acid, salt, hydroxyl, amino or other polar groups present on the
complexing agent molecule can provide a water solubilizing or
hydrophilic effect which is reflected in higher 1n a.sub.w /a.sub.o
values, but due regard must be accorded to the variety of fluids,
organs and tissues in mammals, each of which can have a
distinctively acidic or basic environment, ranging from, for
example, the low pH of the stomach to the relatively high pH of the
intestines. The blood is on the mildly alkaline side at pH = 7.4.
Thus, partition coefficient data on the Tc-complexes of this
invention are preferably obtained throughout the pH range of 5 to
8. The use of partition coefficient data in pharmacology is
well-established; see Andrejus Korolkvas, Essentials of Molecular
Pharmacology, Wiley (interscience), N.Y., N.Y., 1970. It has now
been found that the water/n-octanol system provides useful data for
evaluating lipophilic-hydrophilic balance of Tc-complexes without
in vivo testing. Natural logarithms of partition coefficients are
tabluated in several of the Examples which follow.
Due regard should also be given to chelating effects of some water
solubilizing groups such as COOH (or other acid groups) or OH.
Thus, aromatic mercaptans preferably contain a solubilizing (X)
group meta or para to the SH group in addition to or in lieu of
ortho-OH or ortho-COOH. A solubilizing group substituted on a
second fused or independent aromatic ring serves the same purpose
as the meta or para X group.
When the complexing agents of this invention are combined with an
oxidizable low valence metal salt, the salt can be added to a water
solution of the complexing agent. For example, dihydrothioctic acid
can be dissolved in a sodium bicarbonate-water solution and a
reducing agent comprising an excess over stoichiometry of
SnCl.sub.2.2 H.sub.2 O
dissolved in ethanol can then be added to the solution. After the
complexing and reducing agents have been combined, 99m sodium
pertechnetate can be added. Agitation at a normal ambient
temperature (20.degree.-25.degree.C.) will initiate the reduction
step, and over 50% (in practice, more than 80%) of the
pertechnetate ion will be in reduced form after less than an hour
at this ambient temperature. The extent of reduction can be
determined with thin layer chromatography (T.L.C.) and radiation
monitoring, since TcO.sub.4.sup.- and its reduced-and-complexed
form have distinctly different R.sub.f values if the chromatogram
is developed with properly selected solvents.
If the oxidizable low valence metal salt is omitted, the sodium
pertechnetate eluate can be reacted with HBr to form
H.sub.2.sup.99m TcBr.sub.6. This reaction is preferably carried out
by repeatedly evaporating the eluate in the presence of > 0.1N
(up to concentrated) HBr or using a dry, inert gas such as
nitrogen. The H.sub.2 TcBr.sub.6 can be extracted with acetone,
reacted with an excess of the mercaptan complexing agent in a
non-aqueous medium to form the Tc-complex, and then worked up in
saline solution or the like. Further pH changes can be used, if
necessary to dissolve the Tc complex. The substantially isotonic
radiopharmaceutical is then ready for injection.
The distinct R.sub.f values of novel Tc-mercaptan compounds or
complexes produced according to this invention can reliably
characterize these compounds so that they are distinguished from
their precursors. Since only minute amounts of complexes of
Tc.sup.99m can be produced, analysis of the complex by any method
other than T.L.C. is extremely difficult at best. To reproducibly
determine the R.sub.f values, thin layer chromatographs can be made
from appropriate solutions and a standardized chromatogram sheet.
Reproducible results have been obtained with unactivated 100
micron-thick silica gel chromatogram sheets having a polyvinyl
alcohol binder and a neutral pH. One commercially available form of
such a chromatogram is obtainable from Eastman Kodak Company as
EASTMAN CHROMAGRAM Sheet 6060, described in the references noted in
Kodak Publication Number JJ-7, available from Eastman Kodak
Company.
Several thin layer chromatograms can be made and averaged as a
double check on the experimental error inherent in the R.sub.f, but
generally this error is very small. The chromatograms are developed
with polar solvent systems such as ethanol:water:ammonium
hydroxide, as described subsequently.
The invention is illustrated by the non-limiting Examples which
follow.
EXAMPLE 1
Tc -- Dihydrothioctic Acid, Preparation and Distribution in
Mice
One microliter of DL-6,8-dihydrothioctic acid (hereinafter referred
to as DHT)* was placed in an evacuated N.sub.2 flushed
pharmaceutical vial. One ml of water and 1.3 molar-equivalents
NaHCO.sub.3 (based on eq. of COOH) were added and the sample shaken
vigorously to dissolve the DHT. Twenty-five microliters of absolute
ethyl alcohol containing enough SnCl.sub.2.2 H.sub.2 O to provide
10 micrograms of Sn (II) were added. Four ml of Na.sup.+.sup.99m
TcO.sub.4.sup.- (0.93 millicurie 99m Tc) were added, the vial
vigorously shaken, then allowed to stand for 16 minutes at normal
ambient temperature. Analysis with thin layer chromatography
(T.L.C.) using anhydrous acetone and an EASTMAN CHROMAGRAM 6060
(described subsequently) showed 0.3% unreacted 99m -
pertechnetate.
The solution was diluted to 4 microcuries of 99m Tc per ml and 0.1
ml of this solution was injected, i.v. (intravenously) in the tail
vein of each of seven female Swiss Webster white mice. The mice
were sacrificed at the following time periods: 0, one-half hour,
21/2 hr., 4 hr., 6 hr., and 24 hrs. The organs of each mouse were
isolated and the distribution of 99m Tc determined to assay with a
Packard series 410A Auto-Gamma Spectrometer. The results of this
study are shown in Table IA.
TABLE IA
__________________________________________________________________________
Distribution of 99mTc from Dihydrothioctic Acid -- 99mTc Mercaptide
in Mice as a Function of Time
__________________________________________________________________________
Percent of Total Injected 99mTc as a Function of Time*
__________________________________________________________________________
Organ 0 0.5 hr. 1 hr. 2.5 hrs. 4 hrs. 6 hrs. 24 hrs.
__________________________________________________________________________
Lungs 3.58 0.65 0.41 0.16 0.04 0.05 0.06 Liver 32.2 27.5 19.8 5.46
5.25 2.66 1.36 Spleen 0.10 0.12 0.06 0.00 0.01 0.02 0.07 Kidneys
5.47 2.49 1.02 0.74 0.38 0.52 0.12 Stomach 0.78 0.22 0.13 0.12 0.33
0.07 0.00 Intestines 7.23 16.24 26.49 23.77 6.89 3.90 0.34 Bladder
0.03 0.11 0.04 0.01 0.00 0.00 0.08 Pancreas 0.49 0.07 0.04 0.03
0.01 0.00 0.00 Carcass 28.0 10.3 7.01 1.19 1.42 1.37 1.08
__________________________________________________________________________
*Activity corrected for radioactive decay and counting efficiency
for eac organ. Subsequent studies showed that the percentages in
Table IA, in absolute terms, are subject to a large experimental
error, but nevertheless are very useful as relative values.
The experimental error in the distribution vs. time data for the
99mTc-DHT complex was minimized by averaging six runs under
identical conditions, always with the female Swiss Webster mice.
The effect of organ geometry on radioactivity counting efficiency
was taken into account. Corrections were also made so that, upon
extrapolation back to time zero, the summation of activity in the
organs was equal to the injected activity (i.e. by comparison to
0.1 ml standards). Intestines were assayed as two samples and the
carcass of four samples due to the relatively large volumes of
these samples.
The preparation of the 99mTc-DHT complex was optimized by following
the previously outlined procedure but with the following amounts of
the reactants:
DHT 2.3 mg Stannous ion 20 micrograms NaHCO.sub.3 0.06 millimoles
99mTcO.sub.4 .sup.- solution 4.5 ml, containing 1.0 mc 99mTc.
The reaction was run at ambient temperature for 15 minutes prior to
dilution of the reaction medium to 4 microcuries/ml for injection
into the mice. The results are reported in Table I-B.
TABLE IB
__________________________________________________________________________
Further 99mTc-DHT Studies in Mice Organ Percentage of Total
Injected 99mTc as a Function of Time** 5 min. 0.5 hr. 1 hr. 2 hr. 4
hr. 6 hr. 24 hr.
__________________________________________________________________________
Lungs 1.33 0.70 0.49 0.27 0.06 0.04 0.01 .+-.0.69 .+-.0.14 .+-.0.14
.+-.0.20 .+-.0.06 .+-.0.04 .+-.0.04 Liver 62.9 45.4 27.4 11.8 6.26
4.46 1.64 .+-.5.7 .+-.10.9 .+-.6.0 .+-.4.0 .+-.1.09 .+-.1.16
.+-.0.57 Spleen 0.14 0.09 0.04 0.03 0.01 0.01 0.09 .+-.0.06
.+-.0.04 .+-.0.01 .+-.0.03 .+-.0.01 .+-.0.01 .+-.0.17 Kidneys 5.37
2.76 2.23 1.53 0.89 0.80 0.37 .+-.1.37 .+-.0.24 .+-.0.63 .+-.1.01
.+-.0.29 .+-.0.24 .+-.0.41 Stomach 0.41 0.40 0.34 0.70 0.34 0.63
0.06 .+-.0.47 .+-.0.14 .+-.0.39 .+-.0.73 .+-.0.20 .+-.0.43 .+-.0.09
Intestines 7.37 29.4 35.7 43.9 28.9 19.3 0.83 .+-.0.71 .+-.4.0
.+-.4.8 .+-.7.6 .+-.14.6 .+-.16.9 .+-.0.31 Carcass 21.0 9.83 6.83
4.39 1.47 1.33 1.19 .+-.7.9 .+-.1.89 .+-.1.44 .+-.4.74 .+-.0.83
.+-.1.09 .+-.1.57 Urine and Feces* 1.49 11.3 26.9 37.4 62.1 73.4
95.8
__________________________________________________________________________
*By difference **Average of 6 studies
These studies clearly show the removal of the 99mTc complex from
the blood by the liver and its excretion into the intestine and
finally out in the feces.
T.L.C. analysis of the Tc-dihydrothioctic acid (Tc-DHT) complex was
carried out as follows:
Chromatogram:
Unactivated 100-micron thick silica gel sheet with polyvinyl
alcohol binder, neutral pH (EASTMAN CHROMAGRAM 6060)
Solvent systems:
1. anhydrous acetone
2. ethanol:water:concentrated ammonium hydroxide in the
volume/volume/volume ratio of 95:17:16
Developed chromatograms:
When developed with solvent (1), R.sub.f = 0 for the Tc-DHT
complex, but the R.sub.f was about 1.0 for unreacted
pertechnetate;
When developed with solvent (2), R.sub.f = 0.66 for the Tc-DHT
complex; R.sub.f = 0.75 for unreacted pertechnetate.
Partition coefficients for the Tc-DHT complex of this Example were
determined with a water/n-octyl alcohol system over the pH range of
5 to 8 using radioactive measurements to determine the amount of
Tc-99m in each phase. The expression a.sub.w /a.sub.o is the ratio
of the activity in water to the activity in n-octyl alcohol. For
comparison, partition coefficients over the same pH range were
determined for sodium 99m pertechnetate and cysteine (HS--CH.sub.2
CH(NH.sub.2)COOH). The results are reported in Table II.
TABLE II ______________________________________ Ln of Partition
Coefficients vs. pH .sup.99m Tc-DHT* Com- Na.sup.99m TcO.sub.4
.sup.99mTc.sup.- Cysteine, pH plex,ln(a.sub.w /a.sub.o) ln(a.sub.w
/a.sub.o) ln(a.sub.w /a.sub.o)
______________________________________ 5.0 -1.8 +3.2 +3.7 6.0 +1.2
+3.3 +3.9 7.0 +4.3 +3.5 +4.1 8.0 +7.4 +3.6 +4.3
______________________________________ *DHT =
D,L-6,8-dihydrothioctic acid
The natural logarithms in the above Table are accurate to .+-.0.5.
The 1n(a.sub.w /a.sub.o) curve for the Tc-DHT complex reflects a
marked dependence of partition coefficient upon pH. Although this
invention is not bound by any theory, it is believed that the free
acid (--COOH) form of the DHT-Tc complex is lipophilic and soluble
in cell membranes, while the carboxylic acid salt form is soluble
both in blood and non-biological aqueous media. It is further
theorized that the observed in vivo performance of the DHT-Tc
complex is due in part to the solubility of the free acid form in
cell membranes and the apparent ability of this species to pass
easily from the blood to the bile. Fortunately, this capability
does not appear to detract from the compatibility of the salt form
with aqueous media.
Technetium complexes or species with partition coefficient data
outside the "maximum" range described previously (e.g.
pertechnetate ion and 99mTc-cysteine) have also been studied in
vivo and found to have insufficient liver or gallbladder
specificity to be useful in the preferred type of organ function
studies contemplated by this invention.
EXAMPLE 2
Gamma Ray Monitoring in Anesthetized Dog
To a pharmaceutical vial containing 1.0 microliter of
DL-6,8-dihydrothioctic acid was added 1.0 ml 0.0067 normal
NaHCO.sub.3. The sample was vigorously shaken to dissolve the
dihydrothioctic acid. Twenty-five microliters of absolute ethyl
alcohol containing 10 micrograms Sn (II) as SnCl.sub.2.2 H.sub.2 O
was added. Then 2.31 millicuries Na 99mTcO.sub.4.sup.- eluate in 4
ml saline was added. After 15 minutes at room temperature, a thin
layer chromatogram was run with anhydrous acetone according to the
method outlined in Example 1, and 2.1% unreacted 99m-pertechnetate
was found.
Liver imaging, analysis of liver function, and cholecystography
were carried out by intravenous injection of 412 microcuries in 1.0
ml of this solution into an anesthetized, fasted dog positioned
with its liver under a gamma camera. The output of the gamma camera
was attached to a computer so the data could later be played back,
displayed, photographed and analyzed. At a period of 20 minutes
post-injection, the liver was clearly visualized. As time passed,
the 99mTc could be seen to concentrate in the gallbladder region
until at 150 minutes post-injection, the ratio of activity per 100
cells over the gallbladder region was 12.6 times that over the
liver. After 180 minutes, a gallbladder stimulus comprising 2 dog
units/Kg of cholecystokinin was administered. Within 20 minutes the
activity/100 cells over the gallbladder region had dropped to 33
percent of its value at 150 minutes.
EXAMPLE 3
Part A -- Preparation of 4-Mercapto Butanol
4-mercapto butanol was prepared as follows: ##SPC3##
4.110 gm. CH.sub.2 = CHCH.sub.2 CH.sub.2 OH* plus 4.772 gm.
##SPC4##
were sealed in a 20 ml pharmaceutical vial which was then
evacuated, flushed with oxygen and vigorously shaken. After
approximately 2 minutes, the vial became spontaneously hot. After
the vial cooled, its was analyzed by G.L.C. (gas-liquid
chromatography) and found to have ##SPC5##
present in 87.7% purity.
Twenty ml conc. NH.sub.4 OH was added and the sample was vigorously
shaken. The HS(CH.sub.2).sub.4 OH was extracted with diethyl ether
(Et.sub.2 O) which was dried over CaSO.sub.4 and the ether
evaporated in vacuo. The 4.779 gm. of isolated product was found to
be 87.4% pure 4-mercaptobutanol by G.L.C.
In a second run, the S-acetyl-4-mercaptobutanol was prepared in
98.0% purity (100% recovery) and hydrolyzed to yield
4-mercaptobutanol in 97.9% purity.
Part B -- 4-Mercaptobutanol-99m Tc Mercaptide
One ml of Na.sup.+ 99m TcO.sub.4.sup.- was evaporated three times
with 1 ml 48% HBr under a stream of nitrogen on a steam bath. The
H.sub.2.sup.99m TcBr.sub.6 was extracted with 3 ml acetone. To 0.7
ml of this solution there was added 1 ml 0.03M HS(CH.sub.2).sub.4
OH in acetone.
After 10 minutes at room temperature, 1 ml saline was added and the
acetone removed on a steam bath under a stream of nitrogen. An
additional 1 ml of saline was added and the solution further
diluted with saline to 4 microcurie 99m-Tc/ml. One-tenth ml of this
solution was injected in each of six female Swiss Webster white
mice, each mouse weighing approximately 20 g. The mice were
sacrificed at periods of 0.25, 0.5, 1.0, 2.0, 4.0, and 24.0 hours.
The organs were isolated and the distribution of 99m Tc determined.
To a large extent, the agent was rapidly removed from the blood by
the liver and excreted in the bile into the intestines and
ultimately out in the feces.
The T.L.C. (thin layer chromatography) analysis of the
99m-Tc-4-mercaptobutanol complex was carried out with unactivated,
100-micron thick, silica gel/polyvinyl alcohol, neutral pH
chromatograms (EASTMAN CHROMAGRAM 6060) as in Example 1. The
solvents were: anhydrous acetone and anhydrous methanol. Results
were:
anhy. acetone, R.sub.f = 0 (compared to about 1.0 for
pertechnetate)
anhy. methanol R.sub.f = 0.69 (compared to 0.73 for
pertechnetate)
The a.sub.w /a.sub.o ratios for the 99mTc-4-mercaptobutanol complex
were determined as in Example 1, except that two independently
determined 1n(a.sub.w /a.sub.o) vs. pH plots were averaged. The
results are given in the following Table.
TABLE III ______________________________________ Ln of Partition
Coefficients vs. pH pH ln (a.sub.w /a.sub.o) [average of 2 runs]
______________________________________ 5.0 - 1.1 6.0 - 0.9 7.0 -
0.8 8.0 - 0.7 ______________________________________
These data indicate good lipophilicity and adequate compatibility
with aqueous media throughout the pH range.
EXAMPLE 4
4-Mercaptobutanol-99mTc Complex Distribution in Mice
One ml of 3N HCl, 0.5 ml ethyl alcohol, 90 mg. benzyl alcohol and
45 mg HS(CH.sub.2).sub.4 OH were placed in a 20 ml pharmaceutical
vial which was sealed and put under an atmosphere of N.sub.2. ONe
ml of 99m TcO.sub.4.sup.- eluate and 6.5 ml saline were added. The
sample was heated 10 minutes on the steam bath, then cooled to room
temperature and 3.5 ml of (86.6 mg/ml) sodium acetate in water were
added. The sample was diluted to 4 microcuries/ml and 0.1 ml of
this solution was injected i.v. (intravenous) in each of seven
(approximately 20 gm) female Swiss Webster white mice. At
appropriate time periods the mice were sacrificed, their organs
isolated and assayed for 99m Tc activity in a gamma spectrometer.
The values were corrected for radioactive decay, and counting
efficiency compared to 0.1 ml standards of the solution. This data,
shown in Table II, clearly shows the concentration of 99m Tc in the
liver followed by its excretion into the intestines and out in the
feces and constitutes a useful liver function test.
TABLE IV
__________________________________________________________________________
Distribution of 99m Tc from HS(CH.sub.2).sub.4 OH -- 99m Tc
Mercaptide in Mice as a Function of Time
__________________________________________________________________________
Percent of Total Injected 99m Tc as a Function of Time* Organ 0 0.5
hr. 1 hr. 2 hr. 4 hr. 6 hr. 24 hr.
__________________________________________________________________________
Lungs 4.46 1.07 0.86 0.61 0.41 0.36 0.41 Liver 30.20 23.03 18.40
16.06 11.84 12.19 10.78 Spleen 0.19 0.18 0.14 0.15 0.10 0.11 0.00
Kidneys 7.38 2.51 1.96 1.68 1.18 1.03 0.61 Stomach 0.85 0.86 1.10
0.54 0.86 0.34 1.66 Intestines 10.42 37.90 31.41 18.41 10.93 2.36
10.32 Bladder 0.05 0.04 0.03 0.02 0.02 0.00 0.01 Pancreas 0.77 0.25
0.12 0.23 0.10 0.10 0.00 Carcass 42.35 14.22 10.80 11.56 7.86 6.70
6.74
__________________________________________________________________________
*See Note to Table I in Example 1
EXAMPLE 5
.alpha.-Thio-2-Furan Pyruvic Acid -- 99m Tc Complex
As in previous Example 3, 1 millicurie 99m TcO.sub.4.sup.- in 1 ml
eluate from a generator was evaporated to dryness under a stream of
N.sub.2 three times with 1 ml of 48% HBr. The H.sub.2 TcBr.sub.6
residue was extracted with 3 ml of dry acetone. One ml of this
solution was added to 1 ml of 0.03 molar alpha-thio-2-furan pyruvic
acid* in acetone. After 10 minutes, 1 ml of saline was added, and
the acetone was evaporated under a stream of N.sub.2. One ml of
saline was added plus one drop 2N NaOH to yield pH of 9. The
solution was injected i.v. in the tail vein of six, approximately
20 gram, female Swiss Webster mice. The mice were sacrificed at
appropriate periods of time. The organs were isolated and the
activity distribution determined. This distribution, shown in Table
V, clearly shows the removal of the technetium complex from the
blood by the liver, followed by excretion of the technetium complex
in the bile into the intestines and ultimately excretion in the
feces. This constitutes a liver function test, the kinetics which
can be followed in a human patient for example, by using a gamma
camera or a rectilinear scanner.
TABLE V
__________________________________________________________________________
Distribution of 99m Tc from alpha-thio-2-furan Pyruvic Acid 99m Tc
Mercaptide in Mice as a Function of Time
__________________________________________________________________________
Percent of Total 99m Tc in Mice as a Function of Time* Organ 0.25
hr. 0.5 hr. 1 hr. 2 hr. 4 hr. 24 hr.**
__________________________________________________________________________
Lungs 3.25 1.85 0.76 0.97 0.38 0.26 Liver 48.7 42.4 34.3 31.7 23.9
9.31 Kidneys 4.04 4.34 3.49 3.85 3.13 3.58 Stomach 6.64 8.42 5.89
7.69 2.04 0.00 Intestines 10.8 16.3 26.5 35.9 63.1 1.69 Pancreas
0.84 0.40 0.26 0.34 0.09 1.00 Carcass 25.81 26.2 28.7 19.8 7.16
4.33
__________________________________________________________________________
*These values are not corrected to 0.1 ml standards as in previous
examples, and the footnote to Table I is applicable here also.
**Included urine and feces in total.
The a.sub.w /a.sub.o ratios for the Tc 99m complex of this Example
were determined as in Example 1. The results (1n data accurate to
.+-.0.5) were as follows:
TABLE VI ______________________________________ Ln of Partition
Coefficients vs. pH pH ln(a.sub.w /a.sub.o)
______________________________________ 5.0 + 1.0 6.0 + 1.1 7.0 +
1.3 8.0 + 1.5 ______________________________________
EXAMPLE 6
Partition Coefficients of 99mTc-Aliphatic Mercapto Alcohol
Complexes
A series of TC-99m complexes were made by reducing NaTcO.sub.4 and
reacting the reduced Tc-99m species with the following aliphatic
mercapto alcohols:
2-mercapto ethanol
3-mercapto propanol
5-mercapto pentanol [omega-mercapto-n-amyl alcohol]
4-mercapto-2-methyl-2-butanol
(For the data on the 4-mercaptobutanol complex, see Table III of
Example 3(B).)
The natural logarithms of the partition coefficients at various pH
levels for the water/n-octanol system are set forth in the
following table. Partition coefficients were determined as in
Examples 1, 3, and 5 in Tables II, III, and VI.
TABLE VII ______________________________________ Partition
Coefficients of Tc-99m Complexes Complexing ln a.sub.w /a.sub.o At
Various pH Levels Agent pH=5 pH=6 pH=7 pH=8
______________________________________ 2-mercapto-ethanol - 0.9 -
0.8 - 0.7 - 0.6 3-mercapto propanol - 0.5* - 0.4* - 0.3* - 0.2*
5-mercapto pentanol - 0.2 - 0.1 - 0.1 0.0 4-mercapto- 2-methyl- +
1.0 + 1.1 + 1.2 + 1.3 2-butanol
______________________________________ *Average of two runs
Partition coefficients in the above table have the same level of
accuracy as those of Tables II and VI.
The C.sub.3 and higher mercapto aliphatic alcohols were prepared
from the appropriate unsaturated alcohols by the method of Example
3(A), i.e. by formation and cleavage of the S-acetyl group and
extraction of the product with ether. Thus, 3-mercaptopropanol was
prepared from allyl alcohol, 5-mercaptopentanol from 4-penten-1-ol,
and 4-mercapto-2-methyl-2-butanol from 2-methyl-3-buten-2-ol.
EXAMPLE 7
99mTc-Para-Mercapto-Benzoic Acid 99mTc-2-Mercapto-Ethylamine
A. The compound para-aminobenzoic acid was substituted for
ortho-aminobenzoic acid in the method outlined in Org. Syn.
Collection, Vol. II, page 580. (Para-aminobenzoic acid is
commercially available.) The 99mTc-p-mercaptobenzoic acid complex
was prepared using sodium 99m pertechnetate and HBr, as in Example
3(B). The complex was assayed by intravenous injection into the
tail vein of Swiss Webster mice, as in Examples 1 and 4, the
animals being sacrificed at 0.25, 0.5, 1, 2, 4, and 24 hours. The
assay indicated rapid uptake by the liver and rapid excretion by
the liver into the gut. Uptake by the lungs, kidneys, and stomach
did not detract significantly from liver specificity.
B. The 99mTc complex of the compound 2-mercaptoethylamine was
prepared from reduced sodium 99m pertechnetate. Using the Swiss
Webster mice assay, outlined previously, this complex was found to
be liver specific and rapidly excreted by the liver into the
gut.
* * * * *